Pisep, A Powerful New Ion Exchange Chromatography Using Controlled Ph Gradients For Separating Proteins On Anionic And Cationic Stationary Phases

Abstract

pISep is a new low ionic strength IEX. It consists of externally controlled pH gradients over the pH range from 2 to 12 created by the mixing of two specific cocktails of small organic buffers, one at an acid pH, the other at a basic pH. Gradients can be generated on strong or weak cationic or anionic exchangers over arbitrary pH ranges wherein the stationary phases remain totally charged. Software makes possible the calculation of accurate linear, nonlinear or combined, multi-step, multi-slope pH gradients. An extension of the pISep technology enables the formation of fully controlled, externally generated pH gradients in the presence of additives such as NaCl (up to 1.0 M). The ability to add salt while retaining control over the formation of pH gradients provides much improved flexibility for the separation of proteins sensitive to extremes of pH. Further extensions of the method include the formation of fully controllable pH gradients in the presence of up to 8M urea or 80% acetonitrile. Subsequent creation of mathematical manifolds that define the mixing proportions of the acidic and basic buffers to attain a specific pH as a function of both pH and additive concentration has allowed the creation of software to create simultaneous independent gradients of pH and either salt, urea, or acetonitrile. This latter technology amounts to two dimensional chromatography on a single column. We present examples here of very high resolution separations of model proteins, hemoglobins, MAbs, whole cell extracts, and trypsin digests of BSA , demonstrating the wide versatility of the methodology. Extensions of the electrostatic theory of protein binding to charged stationary phases will also be discussed in light of the experimental results reported here.